We found that the Immunomodulatory Peroxidase (IMPer), an enzyme secreted by the mosquito Anopheles gambiae midgut in response to blood feeding, and dual oxidase (Duox) form a dityrosine network that decreases gut permeability to immune elicitors and protects the microbiota by preventing activation of epithelial immunity. This network provides a permissive environment for Plasmodium development, as it prevents activation of anti-malarial responses mediated by nitric oxide synthase (NOS). When the formation of this barrier is prevented, by silencing either IMPer or Duox, mosquitoes mount strong pathogen-specific responses to bacteria and Plasmodium. Recent experiments indicate that adult mosquito females challenged with Plasmodium respond more efficiently to subsequent challenges. This effect is long lasting and both the priming and the recall require ookinete invasion of midgut epithelial cells in the presence of bacteria from the midgut flora. The priming response involves hemocyte differentiation that results in a long-lasting increase in the number of circulating granulocytes, the major cells involved in phagocytosis. Circulating hemocytes respond to ookinete invasion by attaching to the basal surface of the midgut. We found that this response is enhanced in mosquitoes previously exposed to Plasmodium infection. Furthermore, the transfer of cell-free hemolymph from challenged mosquitoes to newly emerged sugar-fed females induces hemocyte differentiation and confers increased resistance to Plasmodium infection. Our studies indicate that hemocyte differentiation mediates innate immune memory in mosquitoes. The oxidation resistance 1 (OXR1) gene is highly conserved between species and is known to protect yeast, rodent and human cells from oxidative stress through an unknown mechanism. Gene silencing experiments in An. gambiae adult females revealed that OXR1 regulates expression of catalase and glutathione peroxidase, two critical enzymes that detoxify hydrogen peroxide (H2O2);and provided the first experimental evidence that OXR1 expression is regulated by the JNK pathway. OXR1 is essential for efficient reactive oxygen species (ROS) detoxification, as silencing this gene significantly decreases the survival of adult females to an oral challenge with H2O2. OXR1 silencing also dramatically decreases the number of P. berghei ookinetes that survive and transform into oocysts, in agreement with previous studies from our group indicating that ROS are key effectors of mosquito immune responses against bacteria and Plasmodium. The unexpected observation that OXR1 silencing does not affect P. falciparum infection suggests that this parasite species may be less susceptible to ROS damage. We identified a serine protease (SP30) that is required for Plasmodium parasites to efficiently invade midgut epithelial cells. The mechanism by which SP30 silencing affects ookinete midgut invasion is under investigation. SP30 appears to be part of a general signaling cascade triggered by Plasmodium invasion, as SP30 expression is also highly induced when mosquito salivary glands are invaded by Plasmodium sporozoites. Furthermore, SP30 silencing significantly decreases the number of sporozoites that invade the salivary gland. The E. coli expression system was used to produce recombinant SP30, which was purified and injected into rabbits to generate polyclonal antiserum. These antibodies recognize a single band in midguts of sugar-fed females. SP30 protein is highly expressed in the cytoplasm of Plasmodium-invaded cells. The serine protease SRPN6 has been shown to limit Plasmodium invasion. SP30 silencing abolished the induction SRPN6 mRNA in response to Plasmodium midgut infection, suggesting that both genes could be part of the same signaling cascade, with SP30 acting upstream of SRPN6. We are collaborating with Dr. Jose Ribeiro and Dr. Jesus Valenzuela to express a recombinant SP30 that is enzymatically active, to investigate the substrate specificity, enzyme kinetics and the interaction of this enzyme with specific inhibitors. We identified the secreted heme peroxidase (HPX2) that mediates nitration of midgut epithelial cells invaded by Plasmodium. Silencing of HPX2 or NADPH oxidase (Duox) reduces nitration and greatly enhances Plasmdoium survival, supporting the time bomb-model. Furthermore, HPX2 silencing rescues the antiplasmodial effect of NOS induction in IMPer silenced females, indicating that HPX2-mediated nitration is required for NO to have an effective antiplasmodial effect. We identified a QTL in Chr. 13 that confers an African strain of Plasmodium falciparum (GB4) the ability to survive in An. gambiae (L35) females. In contrast, 98-100% of P. falciparum 7G8 parasites (Brazilian strain) are detected by the mosquito immune system and killed. The melanization phenotype in females infected with 7G8 can be completely reversed by silencing genes known to mediate antiplasmodial responses, indicating that these two P. falciparum strains differ in their ability to evade the mosquitos immune system. The QTL encompasses a 171.8 kb region coding for 42 genes. We confirmed that QTL regions using linkage group selection, identified multiple polymorphisms and differences in gene expression. Multiple alternative strategies will be used to identify the gene that confers this phenotype.